Gene expression analysis
Relative gene expression analysis was performed through total RNA
extraction and quantitative real-time PCR (qRT-PCR) analysis following
our previous
operations.30 Briefly,
fresh leaves were ground into powder in liquid nitrogen and total RNA
was extracted using RNA purification kit (Invitrogen Inc., CA, USA).
Following concentration measurement by ultraviolet spectrophotometer
(Cary 50, Varian, USA) and integrity determination by agarose gel
electrophoresis, a reverse transcription procedure was performed with
the M-MLV reverse transcriptase (TaKaRa, Dalian, China) and the resulted
cDNA mixture was used for subsequent PCRs analysis.
The qRT-PCR analysis was carried out in a Rotor-gene-6000 real-time PCR
system (Corbett Research, Mortlake, Australia). The selected genes and
designed primers were listed in Supplementary Table S2. After the
preparation of the reaction mixture containing primers, cDNA and SYBR
Green Master (ROX, Mannheim, Germany), the PCR was performed under the
following temperature procedure: 10 min at 94°C, followed by 40 cycles
of 30 s at 94°C, 30 s at 52–55°C (based on the Tm of the primers) and
20 s at 72°C. The relative abundance of gene expression were determined
by comparative threshold cycle (Ct) method and actin was used as an
internal control.31Three replicates were performed for each sample.